Separation, isolation and concentration are process steps common to a chemical analysis. Often these steps are taken to remove interfering substances so that a subsequent chemical analysis can be performed. This "separation" stage can be performed several ways including solvent extraction, solvent evaporation and resin exchange. Magnetic separation, another technique for removing interfering substances, is a process of separation, isolation and concentration where the sought-for substance is attached or bound to magnetic particles. The magnetic particles offer advantages of handling including speed, convenience and low energy input. It is particularly suited to handling small samples. Advanced Magnetics Inc. of Cambridge, Mass. has been very active in this field in the application of their super paramagnetic particles to separation techniques. Their usage and properties is described in a product bulletin entitled Magnetic Affinity Chromatography Starter Kit M4001 and Magnetic Affinity Chromatography Support Biomag.TM. M4100 dated July 1984.
Magnetic particles are particularly useful in heterogeneous immunoassays as a solid support. To be useful as a solid support, the particles must be derivatized to permit the attachment of bioactive protein. Hersh et al. in their U.S. Pat. No. 3,933,997 describe the use of magnetically responsive particles for this purpose and use functionalized silanes as the intermediate between the particles and the bioactive protein.
There are essentially two types of heterogeneous immunoassays. These are competitive immunoassays and sandwich immunoassays. In a competitive assay, an antibody to an antigen contained in a first reagent is attached to the derivatized magnetic particles to make up a solid phase. The second reagent, consisting of antigen attached to a tag (a measurable entity, including radioactive molecules, fluorescent molecules, or enzymes), and patient sample are mixed with the solid phase in a test tube. In the absence of patient antigen, some 50% of the antigen-tag is bound to the antibody of the magnetic solid phase. In the presence of patient antigen, some of the antibodies are filled up with patient antigen and are unavailable to the tag antigen. As a result increasing amounts of patient antigen leads to decreasing amount of tag antigen. Thus one can form a calibration chart relating the amount of patient antigen to the amount of tag. The separation stage results from the need to measure the free tag or the bound tag, not the total tag added. The magnetic particle facilitates this separation by forming the particles with the bound tag into a pellet on the side of the tube. The free tag can then be removed as by aspiration. Following the separation and removal of free tag, another reagent is added so that the amount of bound tag can be measured. In a typical case, enzyme is used as the tag so that the reagent added is a "substrate" for the enzyme permitting the measurement of the amount of tag that was bound to antibody.
In a typical sandwich immunoassay, an antibody to an antigen is attached to the magnetic particle. This is in high concentration relative to the amount of patient antigen in a sample. Patient antigen is captured by the antibody on the magnetic particles and then the particles (and captured patient antigen) separated from interfering substances in the sample. To this, a second reagent, containing a second antibody with an attached tag, is added. This second antibody attaches to the patient antigen, captured by the first antibody on the magnetic particle, and results in the formation of a sandwich so that the second antibody tag is held firmly by the antigen to the first antibody on the magnetic particle. At this point, a magnetic separation similar to that described, permits the determination of bound tag which is in proportion to the patient antigen, the excess tag of the second reagent having been removed by aspiration.
Magnetic particles are particularly useful as the solid support in heterogeneous immunoassays because they can readily separate the free from the bound tag. Such immunoassays using magnetic particles as a solid support are described for example in U.S. Pat. No. 4,661,408 (Lau et al.), U.S. Pat. No. 4,628,037 issued to Chagnon et al., U.S. Pat. No. 4,672,040 issued to Josephson, and U.S. Pat. No. 4,698,302 issued to Whitehead et al. The methods disclosed in all of these patents relate to manual processes which utilize manual magnetic separation units such as those that are available from Corning Medical, Corning Glass Works, Medfield, Mass. Such manual techniques are relative slow, require relatively strong magnets which are expensive, require considerable manual dexterity, and require an excessive amount of time to effect the separation with the purity required, particularly for sandwich type heterogeneous immunoassay.
Technicon Corporation has offered an automated heterogeneous magnetic immunoassay system for some years. In this system the reagents are combined in a continuous flow process. Having reacted the reagents together, the process then brings the stream through a magnetic field where the magnetic particles are captured and, bound tag measured. The problem with this process is that of continuous flow systems in general. Carryover from one sample to the next tends to produce erroneous results, which error is reduced by reducing the number of samples analyzed per hour.
The Du Pont patented ACMIA technology for digoxin (DGN Method) has been used on the aca.RTM. Discrete Analyzer using resin based column header as solid separation media. This assay has been adapted to run on the Dimension.RTM. Clinical Chemistry System using chromium dioxide magnetic particles as the solid support. Unfortunately for both analyzers, manual treatment of the samples with antibody conjugate reagent (ABC) is required. The Dimension.RTM. assay also involves treatment with chromium dioxide particle reagent (CPR), magnetic separation and transferring of the supernatant to the instrument for photometric measurement. This manual step required is not only time consuming but, because of the manual feature, is subject to error.